Oral pathogen - mediated pro-tumorigenic transformation through disruption of an Adherens Junction - associated RNAi machinery

通过破坏粘附连接相关的 RNAi 机制，口腔病原体介导促肿瘤转化

• 批准号: 10752248
• 负责人: Christina Rachel Kingsley
• 金额: $ 5.07万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2024
• 资助国家: 美国
• 起止时间: 2024-01-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10752248
• 关键词: 3-Dimensional; Adherens Junction; Binding; Biological Markers; Biology; Cancer Patient; Carcinoma; Cell model; Cell-Cell Adhesion; Cells; Chad; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Complex; Confocal Microscopy; Data; Databases; Dental; Development; Diagnosis; Disease; Doctor of Philosophy; E-Cadherin; Epithelial Cells; Epithelium; Fellowship; Fostering; Fusobacterium nucleatum; Future; Homeostasis; Immunofluorescence Immunologic; Immunoprecipitation; In Vitro; Inflammation; Intervention; Knock-out; Knowledge; Link; Malignant Neoplasms; Mediating; Messenger RNA; MicroRNAs; Microbe; Microprocessor; Molecular; Mouth Neoplasms; Oncogenes; Oncogenic; Oral; Oral cavity; Oral mucous membrane structure; Outcome; Pathologist; Pathway interactions; Patients; Periodontal Diseases; Persons; Physiological; Physiology; Protein Analysis; Publishing; Quantitative Reverse Transcriptase PCR; RNA; RNA Interference; RNA analysis; RNA-Induced Silencing Complex; Regulation; Risk; Risk Factors; Sampling; Scientist; Site; Stratified Epithelium; Survival Rate; Testing; The Cancer Genome Atlas; Therapeutic; Tissues; Training; United States; Up-Regulation; Work; cancer risk; cell behavior; cell transformation; crosslink; inflammatory marker; innovation; malignant mouth neoplasm; novel; oral bacteria; oral cavity epithelium; oral pathogen; oral tissue; oral tumorigenesis; pathogen; pathogenic bacteria; periodontopathogen; posttranscriptional; promoter; protein expression; recruit; success; successful intervention; therapeutic RNA; transcriptome sequencing; tumor progression; tumorigenic; two-dimensional

Abstract: Approximately 53,000 people in the United States are diagnosed with oral cancer yearly, and the 5- year survival rate remains poor due to late diagnosis and intervention. Recent evidence has suggested periodontal disease as a potential risk factor, and it is also associated with poor outcomes. Interestingly, periodontal disease and oral cancer share common phenomenon: disruption of epithelial barrier integrity and inflammation. More specifically, the periodontal pathogen Fusobacterium nucleatum has been associated with both barrier disruption and with oral cancer. Still, it is unclear whether disruption of the oral epithelial barrier by Fusobacterium nucleatum is a mechanistic promoter of pro-tumorigenic oral transformation, or just a consequence of it. To answer this question, we interrogate here a cellular complex that is essential for epithelial barrier integrity, called the adherens junction. More specifically, we have found that the adherens junction component PLEKHA7, which is an E-cadherin and p120 binding partner, recruits core components of the RNA interference machinery including the microprocessor, the RNA induced silencing complex, and sets of mRNAs and miRNAs to maintain epithelial homeostasis. PLEKHA7 depletion from cultured epithelial cells results in mis- localization of the RNAi machinery, decreased miRNA silencing activity, increased oncogene expression, and pro-tumorigenic cell transformation. However, such disruption of the RNAi interference machinery, particularly associated with barrier integrity, has not been studied to date in oral cancer, exposing a significant gap in knowledge. We hypothesize that PLEKHA7 recruits and regulates the RNAi machinery at oral epithelial adherens junctions to maintain homeostasis, and that this mechanism is disrupted in the presence of oral pathogens, which in turn promote expression of pro-tumorigenic and inflammatory markers. We will test our hypothesis through two Specific Aims: 1) determine whether oral epithelial adherens junctions recruit the RNAi machinery to regulate function and levels of miRNAs and of their target mRNAs, using two dimensional and three-dimensional epithelial cell models and by examining tissues from patients; 2) determine if the oral periodontal pathogen Fusobacterium nucleatum promotes upregulation of oncogenic and pro-inflammatory markers through miRNA dysregulation, using co-cultures with Fusobacterium nucleatum, immunofluorescence, protein, and RNA analyses. This project is significant, since it will identify a novel molecular mechanism of epithelial pro-tumorigenic transformation in the oral mucosa, advancing our understanding of the disease. The study is innovative, as it links pathogens, oral epithelial barrier function, and localized RNAi and miRNA regulation with oral cancer. We anticipate that the study will be impactful and contribute to future development of effective biomarkers oral cancer, which are critically needed for timely and successful intervention, as well as to the potential development of RNA-based therapeutics. Altogether, this fellowship will provide foundational training in oral epithelial biology to foster the development of the DMD/PhD trainee into a unique and critically needed academic dental scientist.

摘要：美国约有53,000人每年被诊断出患有口腔癌，5-- 由于晚期诊断和干预，年生存率仍然很差。最近的证据表明 牙周疾病是一种潜在的危险因素，并且也与不良预后有关。有趣的是， 牙周疾病和口腔癌具有共同现象：上皮屏障完整性的破坏和 炎。更具体地说，牙周病原体fusobacterium nucleatum与 障碍破坏和口腔癌。尽管如此，目前尚不清楚是否会破坏口腔上皮屏障 核细菌核是促肿瘤的口服转化的机械启动子，或仅仅 结果。要回答这个问题，我们在这里询问一个细胞络合物，这对于上皮必不可少 屏障完整性，称为粘附连接点。更具体地说，我们发现粘附连接处 组件Plekha7是一种E-钙粘蛋白和P120结合伙伴，招募了RNA的核心成分 干扰机制，包括微处理器，RNA诱导的沉默复合物和mRNA集合 和miRNA维持上皮稳态。培养的上皮细胞中的plekha7耗竭导致错误 RNAi机械的定位，miRNA沉默活性降低，癌基因表达增加和 促肿瘤细胞的转化。但是，RNAi干扰机制的这种破坏，特别是 与屏障完整性相关，迄今尚未在口腔癌中进行研究，暴露了很大的差距 知识。我们假设Plekha7招募并调节口服上皮粘附器的RNAi机械 维持体内平衡的结合，这种机制在有口腔病原体的存在下被破坏了， 反过来促进促肿瘤和炎症标志物的表达。我们将通过 两个具体的目的：1）确定口服上皮粘附连接连接是否募集RNAi机械进行调节 使用二维和三维上皮 细胞模型和检查患者的组织； 2）确定口服牙周病原体纤维杆菌是否 Nucleatum通过miRNA失调，促进致癌和促炎性标记的上调， 使用与羟基核，免疫荧光，蛋白质和RNA分析的共培养。这个项目 很重要，因为它将确定上皮促肌融合型转化的新分子机制 口腔粘膜，促进我们对疾病的理解。该研究具有创新性，因为它连接了病原体，口服 上皮屏障功能，以及口腔癌的局部RNAi和miRNA调节。我们预计 研究将具有影响力，并有助于有效的生物标志物口腔癌的未来发展 及时和成功干预以及基于RNA的潜在发展需要至关重要的 疗法。总之，该研究金将提供口腔上皮生物学的基础培训，以促进 将DMD/PhD培训生的开发成一个独特而急需的学术牙科科学家。